Abstract

Abnormal serum urate levels are recognized as a critical factor in the progression of several chronic diseases. To evaluate the antihyperuricemic effect of Davallia formosana, the inhibitory activities of 15 isolated phytochemicals, including five novel compounds of 6,8-dihydroxychromone-7-C-β-D-glucopyranoside (1), 6,8,3′,4′-tetrahydroxyflavanone-7-C-β-D-glucopyranoside (2), 6,8,4′-trihydroxyflavanone-7-C-β-D-glucopyranoside (3), 8-(2-pyrrolidinone-5-yl)-catechin-3-O-β-D-allopyranoside (4), and epiphyllocoumarin-3-O-β-D-allopyranoside (5), were examined against xanthine oxidase (XOD) and in a potassium oxonate-(PTO-) induced acute hyperuricemic mice model. The results indicated that compounds 3 and 5 significantly inhibited XOD activity in vitro and reduced serum uric acid levels in vivo. This is the first report providing new insights into the antihyperuricemic activities of flavonoid glycosides which can possibly be developed into potential hypouricemic agents.

1. Introduction

Hyperuricemia means high levels of uric acid in the blood, a condition considered to be closely associated with increased risks for developing gout, cardiovascular diseases, hypertension, and metabolic syndrome [1, 2]. Xanthine oxidase (XOD) is an important enzyme responsible for the catabolism of purines in humans; it oxidizes hypoxanthine into xanthine and then further forms uric acid [3, 4]. Allopurinol is currently the most effective XOD inhibitor, which is used for treating hyperuricemia and gout by reducing circulating levels of uric acid and vascular oxidative stress [5]. However, serious side effects include skin rashes and allergic reactions that occur in some clinical patients [6, 7].

Recently, several naturally occurring compounds were reported to inhibit XOD activity [8–10]. In particular, plant phenolic compounds, such as phenolic acids and flavonoids, exhibit strong antioxidant activities via scavenging free radicals. Moreover, many studies also indicated that both types of compounds obviously inhibited XOD activity [11–13]. Davallia formosana is a popular herbal medicine used to treat osteoporosis [14]. Several flavan-3-ols, triterpenoids, proanthocyanidins, and mericprocyanidins were isolated from the rhizome of D. formosana [15, 16]. Our preliminary studies revealed that the crude extract of D. formosana rhizomes could inhibit XOD activity. Therefore, in this study, we investigated the constituents of D. formosana and their antihyperuricemic effects. An in vitro XOD-inhibitory assay and in vivo potassium oxonate- (PTO-) induced acute hyperuricemic mouse model were used to evaluate the uric acid-lowering effects of compounds isolated from D. formosana.

2.3. Plant Material

Davallia formosana was collected in Kaohsiung, Taiwan, in July 2010. It was authenticated by Dr. Hsien-Chang Chang (Division of Pharmacognosy, National Laboratories of Food and Drugs, Department of Health, Taiwan). A voucher specimen was deposited at the Department of Medicinal Chemistry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2.5. Determination of XOD-Inhibitory Activity

The inhibitory effect on XOD was determined spectrophotometrically [17]. The reaction mixture consisted of 100 μL of 50 mM potassium phosphate buffer (pH 7.5), 50 μL of 1.5 mM xanthine, 10 μL of sample solution dissolved in dimethyl sulfoxide (DMSO), and 25 μL XOD (0.05 U). The absorption increments at a UV absorbance of 295 nm indicated the formation of uric acid. All determinations were performed in triplicate. Pure compounds and allopurinol for the XO inhibitory activity assays were examined at concentrations of 0, 25, 50, and 100 μM, respectively. The inhibitory activity of XOD was assessed as the inhibitory percent (%) = (1 − b/a) × 100, where “a” is the change in absorbance per minute without the sample, and “b” is the change in absorbance per minute with the sample.

Six-week-old male ICR mice with body weights of about 30.0 g were purchased from BioLASCO (A Charles River Licensee Corp., Yilan, Taiwan). Before the experiments, mice were raised for 1 week to allow them to acclimate to the environment and diet. All mice were given a standard laboratory diet (no. 5001; PMI Nutrition International, Brentwood, MO) and distilled water ad libitum and kept on a 12 h light/dark cycle at 22 ± 2°C. This study was conducted according to institutional guidelines and was approved by the Institutional Animal Care and Utilization Committee (IACUC) of National Taiwan Sport University, Taoyuan, Taiwan. This study was approved by the IACUC ethics committee under protocol IACUC-10004.

2.7. Xanthine Oxidase Molecular Docking

Models of compound 5 in complex with xanthine oxidase were generated through docking compound 5 to the active site of the X-ray crystal structure of bovine xanthine oxidase (PDB id: 3B9J) (Figure 5). In order to predict the position of compound 5 in the active site, we implemented the docking program (GOLD Genetic Optimization for Ligand Docking) (Cambridge Crystallographic Data Center (CCDC), version 3.2) with the Goldscore scoring function. Before docking, the substrate 2-hydroxy-6-methylpurine and all water molecules were removed. The 3D structure of compound 5 was generated and optimized by energy minimization using Discovery Studio v.3.5 (Accelrys Software Inc., USA). GOLD was used to dock compound 5 into the proteins with the flexible docking option turned on. Initially, 500 independent genetic algorithm cycles of computation were carried out with ligand torsion angles varying between −180° and 180°. The search efficiency was set at 200% to ensure the most exhaustive search for the docking conformational space. All other parameters were kept the same as the default settings. Finally, from the 500 docking conformations of compound 5, the top one with the highest GOLD fitness score was chosen to explore the “inhibitor-bond” conformations in the xanthine oxidase active site using Goldscore within the GOLD program. The molecular models of compound 5 were displayed using the PyMOL software (http://www.pymol.org).

2.8. Statistical Analysis

All data are expressed as the mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) was performed with Duncan’s post hoc test for multisample testing. was considered statistically significant.

3. Results and Discussion

3.1. Structure Elucidation

The rhizome of D. formosana was extracted with 80% ethanol and repeatedly chromatographed to obtain 20 compounds. Five new compounds (Figure 1) together with 15 known compounds, including seven flavonoids, six phenolics, and two triterpenoids, were elucidated based on the physical and spectral data.

Figure 1: Chemical structures of new compounds 1–5 isolated from the rhizome of Davallia formosana.

Compound 1 was obtained as a white amorphous powder. The HR-ESI-MS analysis agreed with the molecular formula C15H16O9 ( [M-H]−). The 1H and 13C-NMR data showed typical signals of a flavone nucleus and a glucose unit. Resonances of the flavone moiety were assigned at δ 8.02 (1H, d, Hz, H-2), 6.27 (1H, d, Hz, H-3), and δ 6.50 (1H, s, H-5). The deshielding of the chemical shifts at δ 163.3 and 160.5 indicated a 6,8-dihydroxyl substitution. Glucose signals were determined by 1H-1H COSY and HMQC spectra. The site of the glucose linkage to the flavone was considered to be C-7 from the HMBC experiment. These results suggested that the structure of compound 1 was 6,8-dihydroxychromone-7-C-β-D-glucopyranoside.

Compound 2 was isolated as a white amorphous powder with optical rotation – 43.2°. The negative HR-ESI-MS exhibited quasimolecular ion peaks at 449.1087 [M-H]−. 1H-NMR and 1H-1H COSY spectra showed typical flavanone structural features at δ 5.91 (1H, s, H-5), 5.34 (1H, dd, , 2.8 Hz, H-2), 2.70 (1H, dd, , 3.1 Hz, H-3), and 3.03–3.16 (1H, m, H-3). The coupling constant of 17.9 Hz observed at δ 2.70 indicated that the C-2 substituted aryl group was equatorial. The ABX-type resonance at δ 6.85 (1H, s, H-2′), 6.74 (1H, d, Hz, H-6′), and 6.68 (1H, d, Hz, H-5′) indicated the presence of 1,3,4-trisubstitutions in the B-ring. Additionally, the 1H-NMR spectrum exhibited signals at δ 3.03–4.46 for a sugar moiety. The COSY and HMQC spectra indicated that the sugar moiety was a glucopyranose. The β-configuration of the glucose moiety was determined by the coupling constant of an anomeric proton ( Hz). The resonances of C-6 and C-8 were significantly shifted downfield to δ 166.3 and 162.0, and the HMBC spectrum showed a correlation between the aromatic proton (δ 5.91) and carbonyl carbon (δ 196.9). This evidence indicated that hydroxyl groups were substituted at C-6, 8. Moreover, the HMBC experiment further showed the three-bond correlation between the anomeric proton H-1′′ (δ 4.46) and C-6, 8 (δ 166.3, 162.0), suggesting that the glucose was joined to the A-ring of the aglycone through a C-glycosidic linkage at C-7. Therefore, the structure of 2 was characterized as 6,8,3′,4′-tetrahydroxyflavanone-7-C-β-D-glucopyranoside.

The molecular formula of compound 3 was established as C21H22O10 on the basis of HR-ESI-MS data ( 433.1142 for [M-H]−). The 1H-NMR spectrum of 3 was similar to that of 2, except that the AX-type resonance at δ 6.81 and 7.31 (each 2H, Hz) replaced the ABX-type coupling pattern of compound 2. Six carbon signals at δ 82.9, 80.8, 75.5, 72.9, 72.2, and 63.2 were assigned as a glucopyranose. The orientation of glucose was confirmed to be the β-configuration according to the coupling constant of the anomeric proton ( Hz). The HMBC correlation between glucopyranose H-1′′ and aglycone C-7 suggested that glucose was substituted at C-7 of the aglycone. The molecular weight of 3 lost 16 units compared to that of 2, which supported compound 3 having lost a hydroxyl group at position C-5′. Accordingly, the structure of compound 3 was assigned as 6,8,4′-trihydroxyflavanone-7-C-β-D-glucopyranoside.

Compound 4 was isolated as a yellow amorphous solid. The molecular formula was deduced to be C25H29NO12 from HR-ESI-MS at 534.1628 [M-H]−. According to the 1H and 13C-NMR data, the compound showed a characteristic catechin structure feature at δ 5.07 (1H, d, Hz, H-2), 4.32 (1H, m, H-3), and 2.70 (2H, d, Hz, H-4). The coupling constant ( Hz) confirmed the trans-arrangement of H-2 and H-3. Analysis of 1H-1H COSY and HMQC spectra, the deshielding of the carbonyl signal at δ 181.6, a tertiary carbon signal at δ 50.3 (C-11), and two methylene signals at δ 32.3, 27.0 indicated that a γ-lactam group was contained in the structure. This result was further demonstrated by IR absorption at 1646 cm−1. The long-rang correlation between the signal at δ 5.37 (1H, dd, , 4.6 Hz, H-11) and C-7, 8, 8a suggested that the γ-lactam ring was attached to C-8 (Figure 2). The allopyranose moiety was determined by 1H-1H COSY and HMBC spectra. Additionally, the HMBC correlation between H-3 and C-1′′ demonstrated that the allopyranose residue was linked to C-3 of the aglycone. These results indicated that compound 4 was similar to davallioside A and B [20]. Therefore, compound 4 was a catechin-3-O-β-D-allopyranoside with a γ-lactam substitution at C-8, and it was determined by 8-(2-pyrrolidinone-5-yl)-catechin-3-O-β-D-allopyranoside.

Figure 2: HMBC correlations of compound 4.

Compound 5 was a white amorphous powder, and the molecular formula was determined to be C24H24O12 ( 503.1203 for [M-H]−) by the HR-ESI-MS spectrum. The 1H and 13C-NMR data (Supplementary Tables 1 and 2 available online at http://dx.doi.org/10.1155/2014/873607) showed that compound 5 was comprised of three units, including a flavan-3-ol, a sugar unit, and an α, β-unsaturated carbonyl group. The relative configurations at H-2 and H-3 were assigned as a cis-orientation based on the coupling constant of signals at δ 5.32 (1H, br.s, H-2) and 4.44 (1H, d, Hz, H-3). The methylene protons at δ 2.81 (1H, dd, , 4.3 Hz) and 2.65 (1H, dd, , 5.2 Hz) of H-4 indicated a flavan moiety structure of compound 5. Resonances of four aromatic protons included a typical ABX-type spin system of the 1,3,4-trisubstituted benzene ring [δ 6.95 (brs, H-2′), 6.76 (d, Hz, H-5′), and 6.66 (d, Hz, H-6′)] and one singlet at δ 6.38 attributed to the A-ring of flavan-3-ol. The HMBC correlations between the olefinic proton δ 7.99 (H-10) and C-8 (δ 101.8), as well as δ 6.10 (H-9) and C-7 (δ 154.7), indicated an α, β-unsaturated carbonyl group linked to the C-7, 8 of A-ring. These results revealed that these resonances were consistent with the pyrone ring of a coumarin entity [21]. Acid hydrolysis of 5 yielded epiphyllocoumarin [22] and allopyranose. The β-configuration of the sugar moiety was determined by the coupling constant of the anomeric proton δ 4.51 (1H, d, Hz, H-1′). The HMBC correlation of H-3 to C-1′ demonstrated that the allopyranose residue was linked to C-3 of the aglycone. Therefore, 5 was characterized as epiphyllocoumarin-3-O-β-D-allopyranoside.

3.2. The XOD-Inhibitory Activity of Phytochemicals from D. formosana

The XOD-inhibitory activities of all pure compounds isolated from D. formosana were examined with effects comparable to that of allopurinol (Supplementary Table 3), a clinically used inhibitor, at the same concentration. Compounds 3 and 5 significantly inhibited XOD activity in dose-dependent manners, compared to allopurinol (Figure 3). The IC50 of compounds 3 and 5 was 57.4 and 124.0 μM, respectively, whereas there was no detectable effect for compound 2.

To further confirm the capabilities of compounds 2, 3, and 5 to reduce the uric acid level in vivo, a PTO-induced hyperuricemia mice model was investigated. After 3 h of PTO treatment, the level of serum uric acid had increased to 12 ± 0.14 mg/dL. As shown in Figure 4, PTO-induced serum uric acid levels were reduced by the three test compounds, as well as the reference (allopurinol). At the same concentration (100 mmol/kg), compounds 2, 3, 5, and allopurinol significantly reduced the level of serum uric acid by 33.9%, 41.7%, 46.0%, and 58.1%, respectively, compared to the PTO group (). XOD is an important purine metabolic enzyme, which is a significant target for developing antihyperuricemic drugs. The in vitro and in vivo results suggested that inhibition of XOD activity played an important role in the antihyperuricemic effects of compounds 3 and 5.

Figure 4: The uric acid-lowering effects of compounds 2, 3, and 5 from Davallia formosana on mice with potassium oxonate- (PTO-) induced hyperuricemia. The results are presented as the mean ± SEM (). , , and , compared to the PTO-treated group.

Figure 5: Predicted binding mode of compound 5 docked into the active site of xanthine oxidase. The top and down pictures of each panel display the 3D and 2D structural docking patterns, respectively. The nitrogen and oxygen atoms are shown in dark blue and red colors, respectively. The hydrogen bond formation and the electrostatic interaction between compound 5 (yellow) and the amino acid residues (gray) of XOD are shown in green and light blue dashed lines, respectively.

3.4. Computational Docking Studies of Compound 5

In addition to its uric acid-lowering activity in vivo, compound 5 also inhibits xanthine oxidase activity in vitro. We were interested in visualizing the effects of compound 5 on XOD in order to gain insights into the observed activities. Many studies have shown that flavonoids can inhibit XOD activity via hydrogen bonding and hydrophobic interaction with key amino acid residues on XOD-catalyzed sites such as Arg880, Phe914, Phe1009, and Thr1010 [32]. To determine the preferred positions of binding sites on XOD for compound 5, the 3D model of interaction was analyzed by docking using bovine milk XOD (PDB id: 3B9J)[33].

As shown in Figure 5, the carbonyl group on the benzopyranone forms hydrogen bonds with the active sites, including Arg880 and Thr1010. Furthermore, the 5-hydroxyl group of compound 5 forms hydrogen bonds with Glu802. The docking results show again that the A-ring of compound 5 is sandwiched between Phe914 and Phe1009 and participates in the formation of aromatic interactions (π-π effects) with the two phenylalanines. In addition, three hydrophobic interactions were observed involving the methylene and Pro1076, the 3′,4′-dihydroxyphenyl moiety and Leu1014, and Val1011. It can be seen that binding residues are the same among [34], suggesting that Arg880 and Thr1010 might be of significance for the selective inhibition of XOD by compound 5.

This study obtained five new compounds from D. formosana, and two of them exhibited potent antihyperuricemic activity. Accordingly, our results can provide the scientific basis for development of antihyperuricemic drugs.

Conflict of Interests

All authors declare that they have no conflict of interests.

Authors’ Contribution

Chi-Chang Huang and Keng-Chang Tsai contributed equally to this work.

Acknowledgments

The authors thank Dr. Hsien-Chang Chang for helping identify the plant material. They are grateful to Ms. Shu-Yun Sun (Taipei Regional Analytical Instrumentation Center, NSC) for measurement of the high-resolution ESIMS and Ms. Shwu-Hui Wang (Center for Instrumentation, Taipei Medical University) for measurement of the NMR spectra. They are grateful to the National Center for High-Performance Computing for allowing them to use their computer facilities. This study was supported by a Grant from the National Science Council of Taiwan (NSC101-2320-B-038-012-MY2).